Long-acting VEGF inhibitors for intraocular neovascularization

ABSTRACT

Compositions and methods for treating a VEGF-related ophthalmic disorder in a subject in need comprising, administering intravitreally to the subject a therapeutically effective amount of an anti-VEGF agent, comprising a VEGF binding portion operatively linked to a Fc-IgG, wherein the VEGF binding portion comprises at least one VEGF binding domain that is an IgG-like domain 2 of VEGFR-1.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT Application No.PCT/US2020/061519, filed Nov. 20, 2020, which claims the benefit of U.S.Provisional Patent Application No. 62/939,756, filed on Nov. 25, 2019.The entire contents of which are incorporated by reference herewith.

GOVERNMENT SUPPORT

The invention was made with government support under Grant No. EY031345awarded by National Institutes of Health. The government has certainrights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 19, 2020, isnamed 24978-0595_SL.txt and is 54,480 bytes in size.

TECHNICAL FIELD

The present invention relates to novel long-acting VEGF inhibitors forintraocular neovascularization.

BACKGROUND

The development of a neovascular supply or angiogenesis serves crucialhomeostatic roles since the blood vessels carry nutrients to tissues andorgans and remove catabolic products¹. However, uncontrolled growth ofblood vessels can promote or facilitate numerous disease processes,including tumors and intraocular vascular disorders¹. Although numerousangiogenic factors were initially identified and characterized², workperformed in many laboratories has established VEGF as a key regulatorof normal and pathological angiogenesis as well as vascularpermeability³ ⁴. Alternative exon splicing results in the generation ofmultiple isoforms that differ in their affinity for heparin, includingVEGF₁₂₁, VEGF₁₆₅ and VEGF₁₈₉. VEGF₁₂₁ lacks significant heparin binding.While VEGF₁₆₅ has a single, exon-7 encoded, heparin-binding domain,VEGF₁₈₉ has two heparin-binding domains encoded by exon 6- and exon 7⁵⁶. Much experimental evidence documents the key role of theheparin-binding VEGF isoforms in the establishment of biochemicalgradients required for angiogenesis⁷⁻⁹. VEGF is a member of a genefamily that also includes P1GF, VEGF-B, VEGF-C and VEGF-D. Three relatedreceptor tyrosine kinase (RTKs) have been reported to bind VEGF ligands:VEGFR1¹⁰, VEGFR2¹¹ and VEGFR3¹². VEGF binds both VEGFR1 and VEGFR2,while P1GF and VEGF-B interact selectively with VEGFR1. VEGFR3 bindsVEGF-C and VEGF-D, which are implicated in lymphangiogenesis¹³ ¹⁴ Eachmember of this RTK class has seven immunoglobulin (Ig)-like domains inthe extracellular portion¹⁵. There is agreement that VEGFR-2 is the mainsignaling receptor for VEGF¹⁴, although VEGFR1 binds VEGF withsubstantially higher affinity than VEGFR2¹⁵.

VEGF inhibitors have become a standard of therapy in multiple tumors andhave transformed the treatment of intraocular neovascular disorders suchas the neovascular form of age-related macular degeneration (AMD),proliferative diabetic retinopathy and retinal vein occlusion, which areleading causes of severe vision loss and legal blindness¹⁶ ³ ¹⁷.Currently, three anti-VEGF drugs are widely used in the USA forophthalmological indications: bevacizumab, ranibizumab and aflibercept³.Bevacizumab is a full-length IgG antibody targeting VEGF¹⁸. Even thoughbevacizumab was not developed for ophthalmological indications, it iswidely used off-label due to its low cost. Ranibizumab is anaffinity-matured anti-VEGF Fab¹⁹. Aflibercept is an IgG-Fc fusionprotein²⁰, with elements from VEGFR1 and VEGFR2, that binds VEGF, PIGFand VEGF-B²¹. Importantly, after five-year treatment with ranibizumab orbevacizumab, about half of neovascular AMD patients had good vision,i.e. visual acuity 20/40 or better, an outcome that would have not beenpossible before anti-VEGF agents were available²². However, in real-lifeclinical settings, many patients receive fewer anti-VEGF injections thanin clinical trials and it has been hypothesized that this correlateswith less satisfactory visual outcomes²³. Therefore, there is a need todevelop agents with longer duration after intraocular injection, thusreducing the frequency of injections and a number of approaches to thisend have been attempted²⁴ ²⁵. Aflibercept (EYLEA) was approved based onclinical trials showing that every 8-week administration of the dose of2 mg could match the efficacy of monthly ranibizumab (0.5 mg). However,despite the prediction that a switch to aflibercept would reduce thenumber of intravitreal injections, recent studies suggest that it is notthe case²⁶. Therefore, there is still an unmet medical need forintravitreal anti-VEGF agents with improved half-life.

In 1996, in the course of structure-function studies aiming to identifyVEGF binding elements in VEGFR1, we found that deletion of Ig-likedomain (D) 2, but not of other Ds, abolished VEGF or PIGF binding 27.Replacing D2 of VEGFR3 with VEGFR1 D2 conferred on VEGFR3 the ligandspecificity of VEGFR127. Subsequent studies documented the interactionbetween D2 and VEGF (or P1GF) by X-ray crystallography²⁸⁻³⁰. However, D3was important for optimal VEGF binding²⁷ ²⁸. These initial studies ledto the design of a construct comprising the first three lg-like Ds ofVEGFR1, fused to an Fc-lgG (Flt-1-3-IgG)²⁷. Flt-1-3-lgG showed a potentability to neutralize VEGF in vitro and in several in vivo models ofphysiological and pathological angiogenesis³¹⁻³⁴ ³⁵ ³⁶. However, thehalf-life of this molecule following systemic administration wasrelatively short due to the presence of clusters of basic residues inD3, which resulted in binding to heparan sulfate proteoglycans (HSPG)and sequestration in various tissues.

In 2002 Holash et al²¹ described an IgG fusion construct comprising ofVEGFR1 D2 and VEGFR2 D3, which has much lower heparin-affinity thanVEGFR1 D3. This molecule, known today as aflibercept, ziv-aflibercept orEYLEA, was reported to have a significantly longer systemic half-lifethan Flt-(1-3-lgG)²¹. These PK characteristics, combined with highbinding affinity for VEGF and the ability to bind P1GF and VEGF-B, ledto the prediction that aflibercept would be a more effective anti-tumoragent than other VEGF inhibitors²¹ ³⁷. However, aflibercept has gainedFDA approval only for 2nd line treatment of colorectal cancer, whilebevacizumab and the anti-VEGFR2 antibody ramucirumab received severalFDA approvals in multiple cancer types³ ¹⁷, suggesting that the abovementioned characteristics did not provide a therapeutic advantage.Clearly, aflibercept has had its major clinical impact as anintravitreal treatment for ocular vascular disorders.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for inhibitingangiogenesis and for treating VEGF-associated conditions, such as oculardisease, including but not limited to, age-related macular degeneration,proliferative diabetic retinopathy, retinal vein occlusion, choroidalneovascularization secondary to myopia, retinopathy of prematurity,diabetic macular edema, polypoidal choroidal vasculopathy, comprisingadministering an anti-VEGF agent that inhibits the activity of VEGF and,at the same time, has strong heparin-binding characteristics, therebyproviding superior pharmacokinetics, namely having a longer half-life ofthe therapeutic agent following intravitreal administration.

In embodiments, the present invention provides compositions and methodsfor treating a VEGF-related ophthalmic disorder in a subject in needcomprising, administering intravitreally to the subject a firsttherapeutically effective amount of an anti-VEGF agent, andadministering intravitreally to the subject a second therapeuticallyeffective amount of the anti-VEGF agent within 10 to 30 weeks of theearlier administration. In embodiments, the anti-VEGF agent comprises aVEGF binding portion operatively linked to a Fc-IgG, wherein the VEGFbinding portion comprises at least one VEGF binding domain that is anIgG-like domain 2 of VEGFR-1.

In embodiments, the second therapeutically effective amount of theanti-VEGF agent is administered intravitreally within 16 to 24 weeks ofthe earlier administration. In embodiments, the method comprisessubsequent administrations of the therapeutically effective amount ofthe anti-VEGF agent administered intravitreally within 10 to 30 weeks ofa prior administration for a period of at least one year.

In embodiments, the therapeutically effective amount of the anti-VEGFagent is about 1 to 10 mg. In embodiments, the therapeutically effectiveamount of the anti-VEGF agent is about 3 to 6 mg. In embodiments, thefirst, second and subsequent therapeutically effective amounts are thesame. In embodiments, the first, second and subsequent therapeuticallyeffective amounts are different.

In embodiments, the present invention provides an anti-VEGF agent,wherein the anti-VEGF agent is an Fc-IgG construct fusing domains withVEGF binding characteristics and domains that bind heparinproteoglycans. In embodiments, the present invention provides ananti-VEGF agent, wherein the anti-VEGF agent is an Fc-IgG constructhaving the ability to bind heparin and contains one or more domains withVEGF binding characteristics. In embodiments, the present inventionprovides an anti-VEGF agent, wherein the anti-VEGF agent is a fusionprotein with improved efficacy for binding to VEGF and heparin. Inembodiments, the present invention provides an anti-VEGF agent, whereinthe anti-VEGF agent is a fusion protein with very low endotoxin levels.

In embodiments, the present invention provides an anti-VEGF agent,wherein the anti-VEGF agent is an IgG chimeric protein comprisingelements of VEGF receptors. In embodiments, the present inventionprovides an IgG chimeric protein, wherein the IgG chimeric proteincomprises one or more fragments of the seven immunoglobulin (Ig)-likedomains in the extracellular portion of VEGF tyrosine kinase receptors.In embodiments, the present invention provides an IgG chimeric protein,wherein the IgG chimeric protein comprises one or more extracellulardomain fragments of VEGFR-1 fused with Fc-IgG. In embodiments, thepresent invention provides an IgG chimeric protein comprising at leastone VEGF binding domain VEGFR-1 domain 2 and at least one additionalVEGFR-1 domain 1 or 3, and not including domain 4. In embodiments, thepresent invention provides an IgG chimeric protein, wherein the IgGchimeric protein comprises one or more extracellular domain fragments ofVEGFR-2 fused with Fc-IgG. In embodiments, the present inventionprovides an IgG chimeric protein, wherein the IgG chimeric proteincomprises one or more extracellular domain fragments of VEGFR-1 andVEGFR-2 fused with Fc-IgG.

In embodiments, the present invention provides an anti-VEGF agentcomprising a VEGF binding portion operatively linked to a Fc-IgG,wherein the VEGF binding portion comprises at least one VEGF bindingdomain that is an IgG-like domain 2 of VEGFR-1, and wherein theanti-VEGF agent has a VEGF-stimulated mitogenesis-inhibiting abilitygreater than aflibercept. In embodiments, the present invention providesthat the anti-VEGF agent has a vitreous binding ability greater thanaflibercept. In embodiments, the present invention provides that theanti-VEGF agent has a vitreous bound VEGF-stimulated endothelial cellproliferation-inhibiting ability greater than aflibercept. Inembodiments, the present invention provides that the agent has anincreased half-life in vivo compared to aflibercept.

In embodiments, the present invention provides that the VEGF bindingportion consists essentially of IgG-like domains 1, 2, and 3 of VEGFR-1(V₁₋₂₋₃).

In embodiments, the present invention provides that the VEGF bindingportion consists essentially of IgG-like domains 2 and 3 of VEGFR-1(V₂₋₃).

In embodiments, the present invention provides that the VEGF bindingportion consists essentially of IgG-like domains 1, 2, 3 and 3 ofVEGFR-1 (V₁₋₂₋₃₋₃).

In embodiments, the present invention provides that the VEGF bindingportion consists essentially of IgG-like domains 2, 3 and 3 of VEGFR-1(V₂₋₃₋₃).

In embodiments, the present invention provides pharmaceuticalcompositions comprising a therapeutically effective amount of ananti-VEGF agent as defined claims and a pharmaceutically acceptableexcipient. In embodiments, the present invention provides methods oftreating a VEGF-related disorder in a subject in need comprisingadministering to the subject a therapeutically effective amount of ananti-VEGF agent as defined. The anti-VEGF agent can be directly injectedinto the affected tissue or organ, such as an eye.

In embodiments, the present invention provides a method for treatingocular disease, wherein an anti-VEGF agent is administered locally tothe eye at a dosage corresponding to a molar ratio of 2:1 compared toVEGF. In embodiments, the present invention provides a method fortreating ocular disease, wherein an anti-VEGF agent is administered byintravitreous injection.

In embodiments, the present invention provides a method for treatingocular disease, wherein an anti-VEGF agent is administeredintravitreally once every 10-30 weeks. In embodiments, the anti-VEGFagent is administered intravitreally once every 16 to 24 weeks. Inembodiments, the treatment is continued for a period of at least oneyear.

According to one embodiment, the present invention provides a method fortreating ocular disease comprising administering a therapeuticallyeffective amount of an anti-VEGF agent locally into the eye wherein thetreatment is effective to treat occult, minimally classic, andpredominantly classic forms of wet macular degeneration, wherein theagent is a fusion protein.

In embodiments the invention can be used to treat a wide variety ofVEGF-related disorders including neovascular age related maculardegeneration, choroidal neovascularization secondary to myopia,proliferative diabetic retinopathy, diabetic macular edema, retinalvascular obstruction such as retinal vein occlusion, ocular tumors, vonHippel Lindau syndrome, retinopathy of prematurity, polypoid choroidalvasculopathy, or non-neoplastic disorders that benefit from anti-VEGFtherapy.

According to another aspect, the present invention provides apharmaceutical formulation comprising an anti-VEGF agent in apharmaceutically acceptable carrier formulation for local administrationsuch as into the eye.

In embodiments, the present invention discloses novel constructs,wherein the constructs potently neutralize the activity of VEGF while,at the same time, have strong heparin-binding characteristics.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Immunoglobulin (Ig)-like domain (D) organization of VEGFR1and of the Fc-fusion constructs designed in our study. Red label denotesheparin-binding domain. D2 is an indispensable binding element for VEGFand P1GF, responsible for ligand specificity²⁷. D3 plays an importantrole in binding affinity and stability²⁷ ²⁸ ³⁰. D3 of VEGFR1, but not D3of VEGFR2, is a major heparin-binding site. V23 and aflibercept (EYLEA)differ only in D3, which is from VEGFR2 in aflibercept. D4 is also aheparin-binding site, implicated in receptor dimerization and homotypicinteractions³⁰. Each construct is shown as a monomer for simplicity, butthe recombinant proteins are dimers due the forced dimerization imposedby the Fc.

FIGS. 2A-2B show characterization of purified recombinant proteins. FIG.2A shows silver-stained SDS/PAGE (4-20% Tris) of our purifiedrecombinant fusion proteins and EYLEA. 200 ng of each protein weresubjected to electrophoresis under reducing conditions. Staining wasperformed by SilverQuest Silver Staining kit (Invitrogen). FIG. 2B showsan analytical size-exclusion chromatography (SEC) of V23, V233, V1233and EYLEA, 25 μg of each. The Y-axis represents intensity of absorbance(A280) in milli-absorbance unit, and X-axis represents elution time inminutes.

FIG. 3 shows IC₅₀ values of the inhibitors. Bovine choroidal endothelialcells were maintained as described in Approach For assays, cells areplated at low density. Inhibitors are then added at variousconcentrations as indicated in the figure. VEGF is added at the finalconcentration of 10 ng/ml. Cell densities are evaluated after 5 days.IC50 values were calculated using GraphPad Prism 5 (GraphPad Software,CA). Data shown are based on two independent experiments obtained withhighly purified proteins and are consistent with numerous previousassays.

FIG. 4 shows binding to bovine vitreous.

FIGS. 5A-5D show effects of control IgG, EYLEA or VEGFR1 Fc fusionproteins on laser-induced choroidal neovascularization (CNV) in adultmice. FIG. 5A shows each protein was injected intravitreally in themouse at the dose of 2.5 μg one day before laser treatment. EYLEA wastested also at 25 μg. Asterisks denote significant differences(Student's t test) compared to the appropriate IgG control groups(**p<0.01, *p<0.05). Data are based on three independent experimentswith at least 5 mice per group. Note that the efficacy of EYLEA is inline with the published literature in the same model. FIG. 5B showseffect of the time of injection prior to injury on CNV area. EYLEA atthe dose of 2.5 μg had a significant reduction only when injected at day−1. In contrast, V1233 at the same dose significantly reduced CNV areaeven when injected 7 days or 14 prior to the injection. The left panelshow representative CD31 immunofluorescence images. Asterisks denotesignificant differences (Student's t test) compared to the appropriateIgG control groups (**p<0.01, *p<0.05). n=5. Similar results wereobtained in two independent experiments. FIG. 5C shows V23, V233 andV1233, tested at equimolar doses (4.8 μg of EYLEA and V23, 6.3 μg ofV233 and 7.2 μg of V1233), show greater efficacy compared to EYLEA. Allagents were administered 14 days prior to the laser treatment. Sevendays later, eyes were harvested, and data were analyzed. Asterisksdenote significant differences (Student's t test) compared to theappropriate IgG control groups (**p<0.01, *p<0.05). FIG. 5D shows serumlevels of EYLEA, V23, V233 or V1233 in mice at different time pointsafter intravitreal injection. Each molecule was injected in both eyes inequimolar amounts: 2.4 mg of EYLEA and V23, 3.15 mg of V233 and 3.6 mgof V1233. After 1 day, 3 days, 7 days, 14 days and 21 days, peripheralblood was collected from the tail vein. Human Fc levels were measured byELISA. Values shown are means±SEM. *, p<0.05; **, p<0.01; ***, p<0.001.n=8 per point.

FIGS. 6A-6C show intravitreal injections of V1233 inhibitneovascularization in the OIR model. FIG. 6A shows intravitrealinjections were performed at P7 in C57BL/6j mice using aflibercept,V1233, and control (IgG). A volume of 0.5 pl aflibercept (E) at a doseof 12.5 μg, 2.5 μg, or 1.25 μg versus control IgG at a dose of 3.25 μginjected into the fellow eyes. Littermates were injected with V1233 (3.8μg or 1.65 pg) and control. The concentrations of IgG control,aflibercept 2.5 μg and V1233 3.8 μg were equimolar (see also FIG. 10legend). Animals were then exposed to 75% oxygen from P7 to P12 followedby return to room air. At P17, the animals were perfusion fixed, and theeyes were enucleated, dissected, stained with BSL-FITC and flat mounted.FIG. 6B shows vasoobliteration and neovascularization were analyzedusing automated software as described by Xiao et al. (ref. 116). Thevasoobliterative areas are shown in yellow, and neovascular tufts areshown in red. FIG. 6C shows quantification of neovascularization shows asignificant reduction (p<0.05 t-test with Welch's correction) inneovascularization relative to control with V1233 (3.8 and 1.65 pg) orhigh dose aflibercept (12.5 μg), but not with aflibercept at 2.5 or 1.25μg.

FIG. 7 shows inhibitory effects of fusion protein on BCEC proliferationstimulated by VEGF165 or VEGF121. Results are expressed as % ofinhibition of VEGF-stimulated proliferation relative to control. Cellnumbers were determined by relative fluorescence unit (RFU) 530/590(Excitation/Emission), average of triplicates.

FIG. 8 shows inhibitory effects of recombinant VEGF receptor Fc-fusionproteins on HUVEC proliferation. V123, V1233, V233, V23 or EYLEA(10-2000 ng/ml) was added along with VEGFI65 (10 ng/ml) for 3 days, andcell viability was determined. Results are expressed as % of inhibitionof VEGF-stimulated proliferation relative to control. Cell numbers weredetermined by relative fluorescence unit (RFU) 530/590(Excitation/Emission), average of triplicates. Statistical analysis wasperformed by 2-Way ANOVA in GraphPad Prism software. Statisticalsignificance *p<0.001, **p<0.0001 was calculated by comparing with VEGFalone.

FIG. 9 shows crystal structure of VEGF/VEGFR2 complex (3V2A) wassuperimposed on the crystal structure of the VEGF/VEGFR1 complex (5T89).VEGFR1 residues that can potentially interact with VEGF and that differbetween VEGFR1 and VEGFR2 are labeled. Yellow and blue greyscales: VEGF.Green greyscale: VEGFR1 D2. White: VEGFR1 D3. Analysis points to a moreextensive interaction between VEGF and VEGFR1 D3 compared to VEGFR2 D3.

FIG. 10 shows effects of V1233 on bovine endothelial cell proliferation.Bovine choroidal microvascular endothelial cells (BCECs, VECTechnologies) were seeded in 96-well plates in low glucose DMEMsupplemented with 10% bovine calf serum and incubated with serialdilutions of V1233 (batch1 and batch2) and EYLEA (RegeneronPharmaceuticals) in the presence of 10 ng/ml of hVEGF165 (R&D system).After 5 or 6 days, cells were incubated with Alamar Blue for 4 h.Fluorescence was measured at 530 nm excitation wavelength and 590 nmemission wavelength.

FIG. 11 shows inhibition of VEGF-induced VEGFR2 activation in PromegaVEGF. The Promega VEGF Bioassay (GA2001, Promega) was used to measurethe ability of V1233, to inhibit stimulation induced by VEGF165 inKDR/NFAT-RE HEK293 Cells were incubated with serial dilutions of V1233(batch1 and batch2), EYLEA and Human IgG1 (BE0297, BioXcell) in thepresence of 20 ng/ml of hVEGF165. After a 6-hour incubation, Bio-Glo80Reagent was added, and luminescence was quantified using SpectraMax M5microplate reader. Data were fitted to a 4PLx®curve using GraphPad Prismsoftware.

FIG. 12 shows effects of heparin on VEGFR1 constructs concentrations inCHO cells culture media. Split pool cells (V123, V1233, V233 and V23)into CD FortiCHO media with or without 100 μg/ml heparin (#H3149, Sigma)and incubate at 37° C. with 5% CO2 with humidified atmosphere and 125rpm for 96 hours. The culture media were collected and the expression ofVEGFR1 ECDs was evaluated by ELISA.

FIG. 13 shows CHO-expressed V1233 is fully active in the mouse CNVmodel. 6-8 week male C57/B16 mice were used (n=6). After laserinduction, ˜5 μg of CHO cells-derived and 293 cells-derived V1233 wereinjected intravitreally (1 μl) in each eye. 10 days later,choroid-sclera complex was harvested and fixed. Neovascular area wasindicated by CD31 immunofluorescent whole mount staining. Figure showsthree representative neovascular areas in each group.

FIG. 14 depicts the amino acid sequence and nucleic acid sequence of theentire human IgG1-Fc fragment and VEGFR-1 domain of construct V1-2-3.SEQ ID No: 1 and SEQ ID No: 2, respectively Amino acid sequences andnucleic acid sequences for the IgG-like domains of VEGFR-1 are providedwithin the Figure, as described. The amino acid sequence of V₁ is SEQ IDNo: 15, the amino acid sequence of construct V₂ is SEQ ID No: 16, andthe amino acid sequence of construct V₃ is SEQ ID No: 17.

FIG. 15 depicts the amino acid sequence and nucleic acid sequence of theentire human IgG1-Fc fragment and VEGFR-1 domain of construct V₂₋₃. SEQID No: 3 and SEQ ID No: 4, respectively Amino acid sequences and nucleicacid sequences for the IgG-like domains of VEGFR-1 are provided withinthe Figure, as described. The amino acid sequence of V₂ is SEQ ID No:16, and the amino acid sequence of V₃ is SEQ ID No: 17.

FIG. 16 depicts the amino acid sequence and nucleic acid sequence of theentire human IgG1-Fc fragment and VEGFR-1 domain of construct V₁₋₂₋₃₋₃.SEQ ID No: 5 and SEQ ID No: 6, respectively Amino acid sequences andnucleic acid sequences for the IgG-like domains of VEGFR-1 are providedwithin the Figure, as described. The amino acid sequence of V₁ is SEQ IDNo: 15, the amino acid sequence of V₂ is SEQ ID No: 16, and the aminoacid sequence of V₃ is SEQ ID No: 17.

FIG. 17 depicts the amino acid sequence and nucleic acid sequence of theentire human IgG1-Fc fragment and VEGFR-1 domain of construct V₂₋₃₋₃.SEQ ID No: 7 and SEQ ID No: 8, respectively Amino acid sequences andnucleic acid sequences for the IgG-like domains of VEGFR-1 are providedwithin the Figure, as described. The amino acid sequence of V₂ is SEQ IDNo: 16, and the amino acid sequence of V₃ is SEQ ID No: 17.

FIG. 18 depicts the amino acid sequence and nucleic acid sequence of theentire human IgG1-Fc fragment and VEGFR-1 domain of constructV₁₋₂₋₃₋₃₋₄. SEQ ID No: 9 and SEQ ID No: 10, respectively Amino acidsequences and nucleic acid sequences for the IgG-like domains of VEGFR-1are provided within the Figure, as described. The amino acid sequence ofV₁ is SEQ ID No: 15, the amino acid sequence of V₂ is SEQ ID No: 16, theamino acid sequence of V₃ is SEQ ID No: 17, and the amino acid sequenceof V₄ is SEQ ID No: 18.

FIG. 19 depicts the amino acid sequence and nucleic acid sequence of theentire human IgG1-Fc fragment and VEGFR-1 domain of construct V₂₋₃₋₄.SEQ ID No: 11 and SEQ ID No: 12, respectively. The amino acid sequenceof construct V₂ is SEQ ID No: 16, the amino acid sequence of V₃ is SEQID No: 17, and the amino acid sequence of V₄ is SEQ ID No: 18.

FIG. 20 depicts the amino acid sequence and nucleic acid sequence of theentire human IgG1-Fc fragment and VEGFR-1 domain of construct V₂₋₄. SEQID No: 13 and SEQ ID No: 14, respectively. The amino acid sequence of V₂is SEQ ID No: 16, and the amino acid sequence of V₄ is SEQ ID No: 18.

DETAILED DESCRIPTION

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

Unless defined otherwise, all technical and scientific terms and anyacronyms used herein have the same meanings as commonly understood byone of ordinary skill in the art in the field of the invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice of the present invention, theexemplary methods, devices, and materials are described herein.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, 2^(nd) ed. (Sambrook et al., 1989); OligonucleotideSynthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney,ed., 1987); Methods in Enzymology (Academic Press, Inc.); CurrentProtocols in Molecular Biology (F. M. Ausubel et al., eds., 1987, andperiodic updates); PCR: The Polymerase Chain Reaction (Mullis et al.,eds., 1994); Remington, The Science and Practice of Pharmacy, 20^(th)ed., (Lippincott, Williams & Wilkins 2003), and Remington, The Scienceand Practice of Pharmacy, 22^(th) ed., (Pharmaceutical Press andPhiladelphia College of Pharmacy at University of the Sciences 2012).

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains”, “containing,” “characterizedby,” or any other variation thereof, are intended to encompass anon-exclusive inclusion, subject to any limitation explicitly indicatedotherwise, of the recited components. For example, a fusion protein, apharmaceutical composition, and/or a method that “comprises” a list ofelements (e.g., components, features, or steps) is not necessarilylimited to only those elements (or components or steps), but may includeother elements (or components or steps) not expressly listed or inherentto the fusion protein, pharmaceutical composition and/or method.

As used herein, the transitional phrases “consists of” and “consistingof” exclude any element, step, or component not specified. For example,“consists of” or “consisting of” used in a claim would limit the claimto the components, materials or steps specifically recited in the claimexcept for impurities ordinarily associated therewith (i.e., impuritieswithin a given component). When the phrase “consists of” or “consistingof” appears in a clause of the body of a claim, rather than immediatelyfollowing the preamble, the phrase “consists of” or “consisting of”limits only the elements (or components or steps) set forth in thatclause; other elements (or components) are not excluded from the claimas a whole.

As used herein, the transitional phrases “consists essentially of” and“consisting essentially of” are used to define a fusion protein,pharmaceutical composition, and/or method that includes materials,steps, features, components, or elements, in addition to those literallydisclosed, provided that these additional materials, steps, features,components, or elements do not materially affect the basic and novelcharacteristic(s) of the claimed invention. The term “consistingessentially of” occupies a middle ground between “comprising” and“consisting of”.

When introducing elements of the present invention or the preferredembodiment(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

The term “and/or” when used in a list of two or more items, means thatany one of the listed items can be employed by itself or in combinationwith any one or more of the listed items. For example, the expression “Aand/or B” is intended to mean either or both of A and B, i.e. A alone, Balone or A and B in combination. The expression “A, B and/or C” isintended to mean A alone, B alone, C alone, A and B in combination, Aand C in combination, B and C in combination or A, B, and C incombination.

It is understood that aspects and embodiments of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand embodiments.

It should be understood that the description in range format is merelyfor convenience and brevity and should not be construed as an inflexiblelimitation on the scope of the invention. Accordingly, the descriptionof a range should be considered to have specifically disclosed all thepossible sub-ranges as well as individual numerical values within thatrange. For example, description of a range such as from 1 to 6 should beconsidered to have specifically disclosed sub-ranges such as from 1 to3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc.,as well as individual numbers within that range, for example, 1, 2, 3,4, 5, and 6. This applies regardless of the breadth of the range. Valuesor ranges may be also be expressed herein as “about,” from “about” oneparticular value, and/or to “about” another particular value. When suchvalues or ranges are expressed, other embodiments disclosed include thespecific value recited, from the one particular value, and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another embodiment. It will be furtherunderstood that there are a number of values disclosed therein, and thateach value is also herein disclosed as “about” that particular value inaddition to the value itself. In embodiments, “about” can be used tomean, for example, within 10% of the recited value, within 5% of therecited value, or within 2% of the recited value.

As used herein, “patient” or “subject” means a human or animal subjectto be treated.

As used herein the term “pharmaceutical composition” refers topharmaceutically acceptable compositions, wherein the compositioncomprises a pharmaceutically active agent, and in some embodimentsfurther comprises a pharmaceutically acceptable carrier. In someembodiments, the pharmaceutical composition may be a combination ofpharmaceutically active agents and carriers.

The term “combination” refers to either a fixed combination in onedosage unit form, or a kit of parts for the combined administrationwhere one or more active compounds and a combination partner (e.g.,another drug as explained below, also referred to as “therapeutic agent”or “co-agent”) may be administered independently at the same time orseparately within time intervals. In some circumstances, the combinationpartners show a cooperative, e.g., synergistic effect. The terms“co-administration” or “combined administration” or the like as utilizedherein are meant to encompass administration of the selected combinationpartner to a single subject in need thereof (e.g., a patient), and areintended to include treatment regimens in which the agents are notnecessarily administered by the same route of administration or at thesame time. The term “pharmaceutical combination” as used herein means aproduct that results from the mixing or combining of more than oneactive ingredient and includes both fixed and non-fixed combinations ofthe active ingredients. The term “fixed combination” means that theactive ingredients, e.g., a compound and a combination partner, are bothadministered to a patient simultaneously in the form of a single entityor dosage. The term “non-fixed combination” means that the activeingredients, e.g., a compound and a combination partner, are bothadministered to a patient as separate entities either simultaneously,concurrently or sequentially with no specific time limits, wherein suchadministration provides therapeutically effective levels of the twocompounds in the body of the patient. The latter also applies tococktail therapy, e.g., the administration of three or more activeingredients.

As used herein the term “pharmaceutically acceptable” means approved bya regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopoeia, other generally recognized pharmacopoeia inaddition to other formulations that are safe for use in animals, andmore particularly in humans and/or non-human mammals.

As used herein the term “pharmaceutically acceptable carrier” refers toan excipient, diluent, preservative, solubilizer, emulsifier, adjuvant,and/or vehicle with which demethylation compound(s), is administered.Such carriers may be sterile liquids, such as water and oils, includingthose of petroleum, animal, vegetable or synthetic origin, such aspeanut oil, soybean oil, mineral oil, sesame oil and the like,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents. Antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; and agents forthe adjustment of tonicity such as sodium chloride or dextrose may alsobe a carrier. Methods for producing compositions in combination withcarriers are known to those of skill in the art. In some embodiments,the language “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, isotonic andabsorption delaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. See, e.g., Remington, TheScience and Practice of Pharmacy, 20th ed., (Lippincott, Williams &Wilkins 2003). Except insofar as any conventional media or agent isincompatible with the active compound, such use in the compositions iscontemplated.

As used herein, “therapeutically effective amount” refers to an amountof a pharmaceutically active compound(s) that is sufficient to treat orameliorate, or in some manner reduce the symptoms associated withdiseases and medical conditions. When used with reference to a method,the method is sufficiently effective to treat or ameliorate, or in somemanner reduce the symptoms associated with diseases or conditions. Forexample, an effective amount in reference to diseases is that amountwhich is sufficient to block or prevent onset; or if disease pathologyhas begun, to palliate, ameliorate, stabilize, reverse or slowprogression of the disease, or otherwise reduce pathologicalconsequences of the disease. In any case, an effective amount may begiven in single or divided doses.

As used herein, the terms “treat,” “treatment,” or “treating” embracesat least an amelioration of the symptoms associated with diseases in thepatient, where amelioration is used in a broad sense to refer to atleast a reduction in the magnitude of a parameter, e.g. a symptomassociated with the disease or condition being treated. As such,“treatment” also includes situations where the disease, disorder, orpathological condition, or at least symptoms associated therewith, arecompletely inhibited (e.g. prevented from happening) or stopped (e.g.terminated) such that the patient no longer suffers from the condition,or at least the symptoms that characterize the condition.

As used herein, and unless otherwise specified, the terms “prevent,”“preventing” and “prevention” refer to the prevention of the onset,recurrence or spread of a disease or disorder, or of one or moresymptoms thereof. In certain embodiments, the terms refer to thetreatment with or administration of a compound or dosage form providedherein, with or without one or more other additional active agent(s),prior to the onset of symptoms, particularly to subjects at risk ofdisease or disorders provided herein. The terms encompass the inhibitionor reduction of a symptom of the particular disease. In certainembodiments, subjects with familial history of a disease are potentialcandidates for preventive regimens. In certain embodiments, subjects whohave a history of recurring symptoms are also potential candidates forprevention. In this regard, the term “prevention” may be interchangeablyused with the term “prophylactic treatment.”

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of a compound is an amount sufficient to prevent adisease or disorder, or prevent its recurrence. A prophylacticallyeffective amount of a compound means an amount of therapeutic agent,alone or in combination with one or more other agent(s), which providesa prophylactic benefit in the prevention of the disease. The term“prophylactically effective amount” can encompass an amount thatimproves overall prophylaxis or enhances the prophylactic efficacy ofanother prophylactic agent.

As used herein, the term “therapeutic agent,” “anti-VEGF agent,” “fusionprotein,” “chimeric protein,” or “recombinant protein” comprises a firstpolypeptide operatively linked to a second polypeptide, wherein the“therapeutic agent,” “anti-VEGF agent,” “fusion protein,” “chimericprotein,” or “recombinant protein” inhibits the activity of VEGF.Chimeric proteins may optionally comprise a third, fourth or fifth orother polypeptide operatively linked to a first or second polypeptide.Chimeric proteins may comprise two or more different polypeptides.Chimeric proteins may comprise multiple copies of the same polypeptide.Chimeric proteins may also comprise one or more mutations in one or moreof the polypeptides. Methods for making chimeric proteins are well knownin the art. In some embodiments the term “therapeutic agent,” “fusionprotein,” “chimeric protein,” or “recombinant protein” refers to anyconstructs expressed or synthesized, including but not limited to,peptides or proteins operatively linking one or more of the Ig-likedomains or domain fragments of VEGFR-1 and/or VEGFR-2 with Fc-IgG.

The term “Ig-like domains” refers to Ig-like domains 1-7 of VEGFR-1 andVEGFR-2. The term “Ig-like domain fragments” comprise a portion of afull length domain, generally the heparin and/or VEGF binding orvariable region thereof. Examples of domain fragments include amino acidsequences comprising a segment of at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99% of the full length domain with100% sequence identity and variations thereof. Variations in the aminoacid sequences of fusion proteins are contemplated as being encompassedby the present disclosure, providing that the variations in the aminoacid sequence maintain at least 75%, more preferably at least 80%, 90%,95%, and most preferably 99%. Certain percentages in between areincluded, such as 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%sequence identity. In particular, conservative amino acid replacementsare contemplated. Conservative replacements are those that take placewithin a family of amino acids that are related in their side chains.Genetically encoded amino acids are generally divided into families: (1)acidic amino acids are aspartate, glutamate; (2) basic amino acids arelysine, arginine, histidine; (3) non-polar amino acids are alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan, and (4) uncharged polar amino acids are glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic aminoacids include arginine, asparagine, aspartate, glutamine, glutamate,histidine, lysine, serine, and threonine. The hydrophobic amino acidsinclude alanine, cysteine, isoleucine, leucine, methionine,phenylalanine, proline, tryptophan, tyrosine and valine. Other familiesof amino acids include (i) serine and threonine, which are thealiphatic-hydroxy family; (ii) asparagine and glutamine, which are theamide containing family; (iii) alanine, valine, leucine and isoleucine,which are the aliphatic family; and (iv) phenylalanine, tryptophan, andtyrosine, which are the aromatic family. For example, it is reasonableto expect that an isolated replacement of a leucine with an isoleucineor valine, an aspartate with a glutamate, a threonine with a serine, ora similar replacement of an amino acid with a structurally related aminoacid will not have a major effect on the binding or properties of theresulting molecule, especially if the replacement does not involve anamino acid within a framework site. Whether an amino acid change resultsin a functional fusion protein can readily be determined by assaying thespecific activity of the fusion protein derivative. Fragments or analogsof fusion proteins can be readily prepared by those of ordinary skill inthe art. Preferred amino- and carboxy-termini of fragments or analogsoccur near boundaries of functional domains.

As used herein, an “isolated” or “purified” fusion protein means thefusion protein is the predominant species present (i.e., on a molarbasis it is more abundant than any other individual species in thecomposition), and preferably a substantially purified fraction is acomposition wherein the fusion protein comprises at least about 50% (ona molar basis) of all macromolecular species present. Generally, apurified composition will comprise more than about 80% of allmacromolecular species present in the composition, more preferably morethan about 85%, 90%, 95%, and 99%. Most preferably, the fusion proteinis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

In one aspect the present invention discloses a composition comprising atherapeutic agent, where the therapeutic agent comprises one or moreheparin binding domains of VEGFR-1 or VEGFR-2, and one or more VEGFbinding domains, thereby inhibiting the binding of VEGF to its cognatereceptor. In embodiments, the anti-VEGF agent comprises a VEGF bindingportion operatively linked to a Fc-IgG, wherein the VEGF binding portioncomprises at least one VEGF binding domain that is an IgG-like domain 2of VEGFR-1.

In embodiments, the present invention provides compositions and methodsfor treating a VEGF-related ophthalmic disorder in a subject in needcomprising, administering intravitreally to the subject a firsttherapeutically effective amount of an anti-VEGF agent, andadministering intravitreally to the subject a second therapeuticallyeffective amount of the anti-VEGF agent within more than 8 weeks, orwithin between 10 to 30 weeks of the earlier administration. Inembodiments, the second therapeutically effective amount of theanti-VEGF agent is administered intravitreally within 16 to 24 weeks ofthe earlier administration. In embodiments, the method comprisessubsequent administrations of the therapeutically effective amount ofthe anti-VEGF agent administered intravitreally within 10 to 30 weeks ofa prior administration for a period of at least one year. The inventionprovides such dosing regimens as may be required for any particularindividual subject in need thereof, wherein the second and subsequentadministrations are less frequent than required for an equimolar amountof aflibercept due to a greater heparin binding efficiency thanaflibercept. The invention further provides that following intraocularadministration, plasma levels of the anti-VEGF agents in an individualare lower than plasma levels of aflibercept in an individual followingintraocular administration of an equimolar amount of aflibercept, whichavoids undesirable systemic effects, such as detrimentalneurodevelopmental effects.

In embodiments, the therapeutically effective amount of the anti-VEGFagent is about 1 to 10 mg. In embodiments, the therapeutically effectiveamount of the anti-VEGF agent is about 3 to 6 mg. In embodiments, thefirst, second and subsequent therapeutically effective amounts are thesame. In embodiments, the first, second and subsequent therapeuticallyeffective amounts are different. The invention provides such dosages oftherapeutically effective amounts as may be required for any particularanti-VEGF agent for any particular individual subject in need thereof.

In embodiments, the present invention provides an anti-VEGF agentcomprising a VEGF binding portion operatively linked to a Fc-IgG,wherein the VEGF binding portion comprises at least one VEGF bindingdomain that is an IgG-like domain 2 of VEGFR-1, and wherein theanti-VEGF agent has a VEGF-stimulated mitogenesis-inhibiting abilitygreater than aflibercept. In embodiments, the present invention providesthat the anti-VEGF agent has a vitreous binding ability greater thanaflibercept. In embodiments, the present invention provides that theanti-VEGF agent has a vitreous bound VEGF-stimulated endothelial cellproliferation-inhibiting ability greater than aflibercept. Inembodiments, the present invention provides that the agent has anincreased half-life in vivo compared to aflibercept.

VEGFR VEGF binding domains are well known in the art. Exemplary aminoacid sequences and nucleic acid sequences for the IgG-like domains V₁,V₂, V₃, and V₄ of VEGFR-1 are provided within FIGS. 14-20, respectivelywhich present the entire human IgG1-Fc fragment and VEGFR-1 domains, asdescribed. Amino acid sequences for the individual human IgG-likedomains V₁, V₂, V₃, and V₄ of VEGFR-1 are also individually provided inSEQ ID Nos: 15-18, respectively. The amino acid sequence of V₁ is SEQ IDNo: 15 (the yellow greyscale amino acid sequence in FIG. 14, which iswithin SEQ ID No: 1). The amino acid sequence of V₂ is SEQ ID No: 16(the blue greyscale amino acid sequence in FIG. 14, which is within SEQID No: 1). The amino acid sequence of V₃ is SEQ ID No: 17 (the greygreyscale amino acid sequence in FIG. 14, which is within SEQ ID No: 1).The amino acid sequence of V₄ is SEQ ID No: 18 (the green greyscaleamino acid sequence in FIG. 18, which is within SEQ ID No: 9)

In embodiments, the present invention provides that the VEGF bindingportion consists essentially of IgG-like domains 1, 2, and 3 of VEGFR-1(V₁₋₂₋₃). In embodiments, the anti-VEGF agent comprises amino acidsequence as defined in SEQ ID No: 1.

In embodiments, the present invention provides that the VEGF bindingportion consists essentially of IgG-like domains 2 and 3 of VEGFR-1(V₂₋₃). In embodiments, the anti-VEGF agent comprises amino acidsequence as defined in SEQ ID No: 3.

In embodiments, the present invention provides that the VEGF bindingportion consists essentially of IgG-like domains 1, 2, 3 and 3 ofVEGFR-1 (V₁₋₂₋₃₋₃). In embodiments, the anti-VEGF agent comprises aminoacid sequence as defined in SEQ ID No: 5.

In embodiments, the present invention provides that the VEGF bindingportion consists essentially of IgG-like domains 2, 3 and 3 of VEGFR-1(V₂₋₃₋₃). In embodiments, the anti-VEGF agent comprises amino acidsequence as defined in SEQ ID No: 7.

In embodiments, the invention provides a pharmaceutical composition foruse in treating a VEGF-related ophthalmic disorder in a subject in need,wherein the anti-VEGF agent is as defined herein. In embodiments, thepresent invention provides pharmaceutical compositions comprising atherapeutically effective amount of an anti-VEGF agent as defined claimsand a pharmaceutically acceptable excipient. In embodiments, the presentinvention provides methods of treating a VEGF-related disorder in asubject in need comprising administering to the subject atherapeutically effective amount of an anti-VEGF agent as defined. Theanti-VEGF agent can be directly injected into the affected tissue ororgan, such as an eye.

In embodiments, the present invention provides a method for treatingocular disease, wherein an anti-VEGF agent is administeredintravitreally more than once every 16 weeks. In embodiments, theanti-VEGF agent is administered intravitreally more than once every 16to 24 weeks. In embodiments, the treatment is continued for a period ofat least one year. In embodiments, the therapeutically effective amountof the anti-VEGF agent is about 1 to 10 mg. In embodiments, thetherapeutically effective amount of the anti-VEGF agent is about 3 to 6mg.

In embodiments the invention can be used to treat a wide variety ofVEGF-related disorders including neovascular age related maculardegeneration, choroidal neovascularization secondary to myopia,proliferative diabetic retinopathy, diabetic macular edema, retinalvascular obstruction such as retinal vein occlusion, ocular tumors, vonHippel Lindau syndrome, retinopathy of prematurity, polypoid choroidalvasculopathy, or non-neoplastic disorders that benefit from anti-VEGFtherapy.

In some embodiments, the therapeutic agent is in an administrable dosageform, comprising the therapeutic agent, and an additional excipient,carrier, adjuvant, solvent, or diluent.

In some embodiments, the present invention discloses a pharmaceuticalcomposition suitable for treating and/or preventatively treating asubject, wherein the anti-VEGF agent is contained in an amount effectiveto achieve its intended purpose.

In some embodiments, the therapeutic agent or compositions disclosedherein are administered by injection. In certain embodiments, thecompositions or the therapeutic agent are injected directly into thediseased organ or tissue. In some embodiments, the therapeutic agent canbe topically administered, for example, by patch or direct applicationto the diseased organ or tissue, or by iontophoresis. The therapeuticagents may be provided in sustained release compositions, such as thosedescribed in, for example, U.S. Pat. Nos. 5,672,659 and 5,595,760. Theuse of immediate or sustained release compositions depends on the natureof the condition being treated. If the condition consists of an acute orover-acute disorder, treatment with an immediate release form will bepreferred over a prolonged release composition. Alternatively, forcertain preventative or long-term treatments, a sustained releasedcomposition may be appropriate.

The anti-VEGF agent may also be delivered using an implant, such as butnot limited to an intraocular implant. Such implants may bebiodegradable and/or biocompatible implants, or may be non-biodegradableimplants. The implants may be permeable or impermeable to the activeagent. The specific implants for delivery of the therapeutic agent isdependent on both the affected tissue or organ as well as the nature ofthe condition being treated. The use of such implants is well known inthe art.

The anti-VEGF agent described in this invention can be formulated innanoparticles or other drug formulations in order to provide precisedelivery to specific tissues and also provide controlled releasetherapy.

The anti-VEGF agent described in this application can be delivered notonly as purified recombinant proteins but also by a gene therapyapproach. Recombinant adeno-associated vectors (rAAVs) or other suitablevectors can be used to deliver the VEGF inhibitor by sub-retinal orintravitreal delivery^(43,44).

In a related aspect, the present invention provides a method fortreating a VEGF-related or neovascular disorder in a subject, whereinthe method involves administering to the subject: (a) an effectiveamount of a fusion protein capable of binding heparin and diminishing orpreventing the development of unwanted neovasculature. The fusionprotein may be combined with other anti-VEGF agents including, but arenot limited to: antibodies or antibody fragments specific to VEGF;antibodies specific to VEGF receptors; compounds that inhibit, regulate,and/or modulate tyrosine kinase signal transduction; VEGF polypeptides;oligonucleotides that inhibit VEGF expression at the nucleic acid level,for example antisense RNAs; and various organic compounds and otheragents with angiogenesis inhibiting activity.

The invention provides that heparin-binding mediated by D3 (or otherIg-like domain) of VEGFR1²⁸, while a disadvantage for systemicadministration, can confer important advantages for intravitreal (orother local) administration. Indeed, the ability to bind HGPSG, keycomponents of the extracellular matrix²⁹, promotes accumulation in thevitreous as well as retinal penetration³⁰. The invention provides aseries of VEGFR-1 Fc fusion constructs having differential abilities tointeract with HSPGs. This enables election of VEGF inhibitors withdifferent duration/half-life in the eye, which are useful underdifference clinical conditions.

The features and other details of the invention will now be moreparticularly described and pointed out in the following examplesdescribing preferred techniques and experimental results. The examplesare provided for the purpose of illustrating the invention and shouldnot be construed as limiting.

EXAMPLES

To identify more effective and longer-lasting VEGF inhibitors forintraocular use, the diversity of heparin-binding in VEGFR1 Ds wasstudied. To this end, eight VEGFR1-Fc fusion constructs were designedhaving differential heparin binding, thus providing a spectrum of HPSGaffinity. FIG. 1 illustrates the domain structure of these protein andhighlights heparin-binding domains. All proteins include D2, the keydeterminant of ligand specificity²⁷. Two constructs (V1233 and V233)have a duplicated D3. The domain structure of aflibercept is also shown.

In initial experiments, the expression levels of several of constructswere low; V1234, V1233, V234 and V124 were detectable at low levels inthe conditioned media. Interestingly, earlier studies had shown thatVEGF isoforms with high affinity for heparin (VEGF189 or VEGF206) arealmost undetectable in the conditioned media of transfected cells, beinglargely bound to the cells surface or the extracellular matrix³⁸ ⁹.However, they could be released in a soluble form by the addition ofheparin or heparinase, indicating that the binding site consisted ofHSPG³⁸ ⁹. Thus, it was determined whether the addition of heparin mayalso affect the levels of recombinant VEGFR-1 fusion proteins. Indeed,adding heparin to the media of transfected cells resulted indose-dependent increases in the concentrations of recombinant protein inthe medium (data not shown).

Purification of the recombinant proteins simply by conventional proteinA (PA) affinity chromatography was attempted. However, this methodyielded a major band of the expected mass and numerous other minorbands, likely reflecting the interaction of the strongly basic,heparin-binding, recombinant proteins with host cell-derived HSPGs andother anionic molecules. Therefore, a protocol was developed thatremoved such impurities, as described in Methods. A wash at high pH(9.2), in the presence of 1.2 M NaCl, while the protein is bound to PA,resulted in release of numerous contaminants. The next step, anionexchange chromatography, was very effective at removing the bulk ofcontaminants and aggregates, while the purified protein was in theflow-through. The LPS levels in the final purified preparations were<0.1 EU/mg (range 0.02-0.08), a very low level compatible withpreclinical studies³⁹. As shown in FIG. 2A, the purity of therecombinant proteins was >95%, as assessed by silver-stained SDS/PAGEand was similar to that of the FDA-approved drug EYLEA. FIG. 2B showsanalytical SEC profiles of the three most promising candidates, V23,V1233 and V233, next to EYLEA. Similar to EYLEA, the three proteinseluted as a single peak at the expected retention time, withoutsignificant aggregation.

The recombinant proteins were tested for their ability to inhibitmitogenesis induced by VEGF165 (10 ng/ml) in BCEC. As illustrated inFig., they had inhibitory effects, with IC₅₀ values were in the range of˜1 nM, except for V124 and V24, which were less potent (FIG. 3). We alsodocumented their ability to inhibit BCEC mitogenesis stimulated byVEGF121 (FIG. 7). Interestingly, EYLEA, in nearly all experimentsperformed (>10) was potent, being active at low concentrations, withIC₅₀ of ˜1 nM, but inhibited no more than ˜80% of VEGF-stimulatedproliferation even at the highest concentrations tested. Similar resultswere obtained using HUVEC proliferation assays (FIG. 8). In contrast,the VEGFR1 constructs, (except, V124 and V24), completely blockedVEGF-induced proliferation. The ability to detect such differenceslikely reflects the relatively high dynamic range of our BCECproliferation assay in response to VEGF stimulation (˜4-fold increase).VEGFR1 D3 may provide a better interactive surface than D3 from VEGFR2,especially considering that VEGFR1 binds VEGF significantly moreeffectively than VEGFR2⁴⁰ ⁴¹ To test this hypothesis, a comparison wasperformed of Protein Data Bank Files of the VEGFR1/VEGF complex (5T89)³⁰and VEGFR2/VEGF complex (3V2A)⁴² and superimposed D2-D3 from eachreceptor. This analysis supports the hypothesis. For example, Arg280 inVEGFR1-D3 interacts with the sidechain of VEGF Phe36, whereas VEGFR2 hasan Asp there. Likewise, in VEGFR1 both Arg261 and Asn290 interact withVEGF Glu64; in VEGFR2 the Arg261 is replaced by Gly and hence in VEGFR2only the Lys replacing Asn290 can interact with VEGF Glu64. FIG. 9illustrates the VEGFR1 residues that can potentially interact with VEGFand that differ between VEGFR1 and VEGFR2.

To further define therapeutically relevant interactions, it was assessedwhether the recombinant proteins bind bovine vitreous in vitro. Asillustrated in FIG. 4, while EYLEA, control IgG or bevacizumab hadlittle or no binding, our proteins showed significant binding. Thestrongest binders were V1233, V233 and V1234, followed by V123. V23 hadintermediate binding characteristics, between EYLEA (or control IgG) andV1233. Vitreous binding was displaced by heparin in a dose-dependentmanner.

Recombinant proteins were tested in the mouse CNV model and compared tocontrol IgG or EYLEA. An extensive literature documents the ability ofanti-VEGF agents to suppress neovascularization in this model⁴³ ⁴⁴ ⁴⁵.Relatively low doses were chosen for proof-of-concept studies, beingbest suited to reveal potency and durability differences among thevarious proteins. Also, it has been reported that intravitrealadministration of high doses of antibodies of the IgG1 isotype may haveoff-target angio-inhibitory effects, mediated by Fc signaling throughFcgRI and c-Cbl, leading to impaired macrophage migration⁴⁶. Theseeffects might potentially confound the interpretation of the data. Thedoses employed are efficacious and at the same time should avoid suchoff-target effects. Initially, each protein was injected intravitreallyat the dose of 2.5 μg one day before laser treatment. EYLEA was testedalso at 25 μg. As illustrated in FIG. 5A, EYLEA resulted in anapproximately 30% inhibition at the dose of 2.5 μg and ˜50% inhibitionat the dose of 25 μg. These findings are largely consistent with thepublished literature. For example, Saishin et al. reported that theintravitreal injection of ˜5 μg of aflibercept resulted in ˜30%inhibition of CNV area in the mouse⁴⁴. Indeed, the dose of 40 μg iscommonly used to achieve maximal inhibitory effects of aflibercept inthe mouse CNV model⁴⁷.

An unexpected finding was the greater potency of some of the constructs:V123, V23, V1233 and V233. Administering 2.5 μg of these proteins, oneday before the injury, matched or even exceeded the level of inhibitionachieved with 25 μg of EYLEA. However, none of the constructs thatincluded D4 demonstrated significant CNV inhibition (FIG. 5A).

To determine whether heparin-binding may translate in durabletherapeutic effects following a single administration, V1233, EYLEA orcontrol IgG, were injected intravitreally (2.5 μg) 1 day, 7 days or 14days before the laser-induced injury. As shown in FIG. 5B, EYLEAresulted in a significant inhibition only when administered 1 day beforethe injury. In contrast, V1233 resulted in a significant inhibition alsowhen administered 7 days or 14 days prior to the injury.

In a subsequent study, equimolar amounts of EYLEA, V23, V1233 and V233,(4.8 μg of EYLEA and V23, 6.3 μg of V233 and 7.2 μg of V1233) wereadministered 14 days prior to the injury. FIG. 5C shows that, at thedose tested, EYLEA had very little effect on CNV. In contrast, V23,V1233 and V233 resulted in a significant CNV inhibition. A prediction ofthe hypothesis is that inhibitors with strong heparin-bindingcharacteristics will have lower systemic exposure compared to EYLEA.Both eyes were injected intravitreally with equimolar amounts of EYLEA,V23, V233 or V1233 and human Fc serum levels were measured at differenttime points up to 21 days after intravitreal administration, as shown inFIG. 5D. EYLEA administration resulted in the highest serum levelsthroughout the experiment. V23, which has a single heparin bindingdomain, resulted in lower serum levels than EYLEA, but trended higherthan V1233 or V233.

Finally, we compared multiple doses of V1233 and EYLEA in the OIR model.In agreement with the findings in the CNV model, V1233 was more potentthan EYLEA at inhibiting neovascularization FIG. 6.

FIG. 7 shows inhibitory effects of fusion protein on BCEC proliferationstimulated by VEGF165 or VEGF121. Results are expressed as % ofinhibition of VEGF-stimulated proliferation relative to control. Cellnumbers were determined by relative fluorescence unit (RFU) 530/590(Excitation/Emission), average of triplicates.

FIG. 8 shows inhibitory effects of recombinant VEGF receptor Fc-fusionproteins on HUVEC proliferation. V123, V1233, V233, V23 or EYLEA(10-2000 ng/ml) was added along with VEGFI65 (10 ng/ml) for 3 days, andcell viability was determined. Results are expressed as % of inhibitionof VEGF-stimulated proliferation relative to control. Cell numbers weredetermined by relative fluorescence unit (RFU) 530/590(Excitation/Emission), average of triplicates. Statistical analysis wasperformed by 2-Way ANOVA in GraphPad Prism software. Statisticalsignificance *p<0.001, **p<0.0001 was calculated by comparing with VEGFalone.

FIG. 9 shows crystal structure of VEGF/VEGFR2 complex (3V2A) wassuperimposed on the crystal structure of the VEGF/VEGFR1 complex (5T89).VEGFR1 residues that can potentially interact with VEGF and that differbetween VEGFR1 and VEGFR2 are labeled. Yellow and blue greyscales: VEGF.Green greyscale: VEGFR1 D2. White: VEGFR1 D3. Analysis points to a moreextensive interaction between VEGF and VEGFR1 D3 compared to VEGFR2 D3.

The activity of purified CHO-expressed V1233 was tested in twoindependent bioassays: BCEC proliferation (FIG. 10) and the Promega VEGFBioassays (FIG. 11). Both assays show that two independent batches ofpurified V1233 inhibit VEGF-stimulated growth or receptor activationwith similar (if not greater) potency as EYLEA.

It was also determined that, in contrast to 293 cells (Expi-293 system),in CHO cells expression of the constructs is not dependent on theaddition of heparin to the medium (FIG. 12), a considerable advantage.In addition, we determined that CHO-derived V1233 is fully active in themouse CNV model and is no less potent than 293 expressed V1233 (FIG.13).

Discussion

Interaction of D3 with the HPSG has been long considered a limitation ofVEGFR1-based anti-VEGF strategies due to sequestration in varioustissues, resulting in reduced systemic half-life. To overcome suchissue, Holash et al. replaced VEGFR1 D3 with VEGFR2 D3²¹. To the sameaim, Lee et al. more recently introduced a glycosylation site in VEGFR1D3, effectively neutralizing positive charges and thus eliminatingD3-mediated HSPG binding⁴⁸. In both cases, systemic half-life wasincreased relative to the original VEGFR1 construct²¹ ⁴⁸.

The present study designed a series of VEGFR-1 Fc fusion constructshaving differential abilities to interact with HSPGs. The premise wasthat heparin-binding, mediated by VEGFR1 D3 (or other Ig-like D such asD4⁴⁹), while a disadvantage for systemic treatment, might confer uniqueadvantages on a VEGF inhibitor to be used for intravitrealadministration, since a) it should anchor the inhibitors to HPSGs orother anionic molecules in the vitreous or other structure in the eye,thus increasing its half-life; b) such inhibitor does not need to beuniformly distributed or to deeply penetrate into the eye structures inorder to effectively bind and block VEGF. A variety of studies haveshown that VEGF can diffuse to a considerable distance from itsproduction site in response to biochemical gradients determined by HPSGor receptor distributions in the vasculature or other sites⁵⁰ ⁸ ⁹. Forexample, although VEGF is produced by tumor cells even at a significantdistance from the vasculature, it diffuses and accumulates in the bloodvessels by virtue of its high affinity for the VEGF receptors,⁵¹⁻⁵³.Therefore, vitreous-bound VEGFR1 variants are expected to generatestrong gradients, capable of attracting and neutralizing VEGF.

Given the challenges in obtaining accurate affinity measurements usingsensor platforms such as SPR with very tight binders (Kd<100 pm)⁵⁴, theconflicting data regarding the affinity of aflibercept versus other VEGFinhibitors²¹ ⁵⁵ and the poor correlation between binding affinity andtherapeutic potency/efficacy among neutralizing antibodies to VEGF andother targets⁵⁶ ⁵⁷, this study chose to focus on biological IC₅₀ data,being more physiologically relevant. As illustrated in FIG. 4, therecombinant proteins had inhibitory effects, with IC₅₀ values in therange of ˜1 nM, except V124 and V24, which were significantly lesspotent.

These proteins bind to bovine vitreous. The strongest binders were,V1233, V1234, followed by V123. V23 had significant but lower vitreousbinding. Control IgG, EYLEA, or AVASTIN had instead minimal binding.

An unexpected finding of our study was the greater potency of some ofthe constructs: V123, V23, V1233 and V233. Administering 2.5 μg of theseconstructs one day before the injury matched or even exceeded the levelof inhibition achieved with 25 μg of EYLEA. The finding that V1233, butnot EYLEA, has significant effect in preventing CNV when administered 7days or 14 days before the injury, documents the durability of theeffects and the therapeutic value.

Also, it was found that intravitreal injection of these heparin-bindingproteins results in much lower systemic levels than EYLEA. This propertymight be particularly useful, for example, for the treatment of ROP,since it has been reported that treatment with anti-VEGF agents withsignificant systemic exposure may have detrimental neurodevelopmentaleffects⁵⁸ ⁵⁹.

Interestingly, none of the constructs containing D4 (V1234, V234, V124,V24) resulted in marked inhibition in vivo (at least at the dosetested), in spite of the fact that these molecules (with the exceptionof V₂₋₄) demonstrated an ability to inhibit VEGF-stimulated mitogenesisin vitro. However, all of these constructs demonstrated a propensity toform oligomers or aggregates, as assessed by SDS/PAGE under non-reducingconditions and size exclusion chromatography (data not shown). Althoughearlier work⁶⁰ identified D4 (together with D7) as a requirement forVEGFR-1 dimerization, such effect has been known to be ligand-dependent.Crystal structure studies revealed a loop in D4 responsible for suchhomotypic interactions³⁹. It is conceivable that high concentrationsand/or the forced dimerization imposed by the Fc construct may result inligand-independent interactions, resulting in aggregation. In any event,aggregates are not desirable pharmaceuticals given the possibility ofinflammation and immunogenicity^(61, 62). Importantly, the lack ofsignificant efficacy of our D4-including proteins argues against thepossibility that a contaminant may be responsible for the observedefficacy, since all proteins were purified by the same methodology andhave strong heparin-binding properties.

In conclusion, aflibercept was designed to eliminate the heparin-bindingheparin domain in order to improve systemic half-life for oncologicalindications. The constructs described in the present study are insteaddesigned to promote binding and retention in the vitreous to ensure moresustained and therapeutically relevant interactions.

In experiments in which CHO cells were employed as an expression system,the requirement for adding heparin to the media of transfected cells wasgreatly diminished, such that adding heparin to the media resulted invery small increases in the recombinant protein concentrations. This islikely explained by differences in HSPG composition/concentrationsbetween 293 and CHO cells.

Methods

For construction of VEGFR-Fc expression plasmids, the nucleic acidfragments encoding the signal peptide and a combination of extracellularIg-like domains one to four of VEGRF127 (Gene ID: 2321) were synthesizedby GenScript USA Inc. The following constructs were done: V123, D1, D2and D3; V23, D2 and D3; V1233, D1, D2, D3 and D3; V233 D2, D3 and D3;V1234, D1, D2, D3 and D4; V234, D2, D3 and D4; V124, D1, D2 and D4; V24,D2 and D4. The synthesized fragments were inserted into pFUSE-hIgGl-Fc1vector (InvivoGen, #pfuse-hgifc1) at EcoRI and BgIII sites, generatingthe plasmids containing the various VEGFR1 ECDs. Then, using PrimeSTARMutagenesis Basal Kit (Takara, R046A), the interval amino acids R and S(BgIII site) between the ECDs and the Fc fragment were removed,generating the plasmids expressing the fusion proteins of VEGFR1 ECDswith a 227-amino acid human IgG1-Fc.

Transfection and Conditioned Media Preparation

The Expi293 expression system (Life technologies, A14524) was used togenerate the conditioned media for purification, according to themanufacturer's instructions. In brief, Expi293F™ Cells (ThermoFisher)were suspension-cultured in Expi293™ expression medium at 37° C. in ahumidified atmosphere with 8% CO2. When the cell density reached to 2.5million/ml, plasmids DNA and ExpiFectamine™ 293 reagent was mixed,incubated 5 min and added to the cells. The final concentration of theDNA and transfected reagent was 1 μg and 2.7 μl per milliliterrespectively. Five hours after transfection, 100 μg/ml Heparin (Sigma,H3149) and protease inhibitor cocktail, 1:400 (Sigma, P1860), were addedto the cells. 16 hours after transfection, enhancer reagents 1 and 2were added. Ninety-six hours after transfection, conditioned media wereharvested. Aliquots were tested for Fc fusion proteins concentrationsusing a human Fc ELISA Kit (Syd Labs, EK000095-HUFC-2) according to themanufacturer's instructions. Protease inhibitors were added (1:500) tothe bulk, which was stored at −80° C. until further use.

Purification of Recombinant Proteins

Pyrogen-free reagents were employed. Prior to use, columns and equipment(Akta Explorer System) were sanitized by exposure to 0.5 N NaOH.Conditioned media from transfected cells were adjusted to PBS, 0.01%polysorbate (PS) 20. PS20 was added to buffers at all steps. Aftercentrifugation at 20,000×g for 30 minutes, supernatants were subjectedto protein A (PA) affinity chromatography using a Hi-Trap MabSelect SuRe(5 ml, GE Healthcare). After loading, the column was washed with 20 mMdiethanolamine, pH 9.2, 1.2 M NaCl, prior to elution with 0.1 M citricacid, pH 3.0, which was immediately neutralized. The PA elution pool wasthen diluted in 20 mM diethanolamine, pH 9.2, and applied to Hi-Trap Q(GE Healthcare) anion-exchange column. The bound material was elutedwith a gradient of NaCl. The flow-through, which contained the purifiedrecombinant protein, was immediately adjusted to 20 mM Tris, pH 6.8, andthen concentrated through binding to heparin-sepharose (Hi-Trap™-HS).After a wash with 0.2-0.45 M NaCl (depending on the construct), therecombinant VEGFR1 fusion protein was eluted with 1 M NaCl. The finalpolishing step consisted of size-exclusion chromatography (SEC).Finally, the proteins were buffer-exchanged by dialysis into 10 mM Tris,pH 6.8, 10 mM histidine, 5-7% threalose, 40 mM NaCl, 0.01% PS20. Thegoal is obtaining a close to iso-osmolar formulation (˜300 mOsm). Todetermine endotoxin levels, ToxinSensor Chromogenic LAL Endotoxin AssayKit (GenScript, L00350) was used according to the manufacturer'sprotocol.

Cell Proliferation Assays

Endothelial cell proliferation assays were performed essentially aspreviously described⁶³ ⁶⁴. Primary bovine choroidal endothelial cells(BCEC) (passage <10) (VEC Technologies Rensselaer, N.Y., Cat #BCME-4)were trypsinized, re-suspended and seeded in 96-well plates (no coating)in low glucose DMEM supplemented with 10% bovine calf serum, 2 mMglutamine, and antibiotics, at a density of 1000 cells per well in 200μl volume. rhVEGF₁₆₅ (R& D Systems, Cat #293-VE-010) or rhVEGF₁₂₁ (R& DSystems Cat. #4644-VS010) was added at the concentration of 10 ng/ml.Aflibercept (EYLEA) was purchased from a pharmacy. The inhibitors wereadded to cell at various concentrations, as indicated in the figures.,before adding the ligands. After 5 or 6 days, cells were incubated withAlamar Blue for 4 hr. Fluorescence was measured at 530 nm excitationwavelength and 590 nm emission wavelength.

Primary human umbilical vein endothelial cells (HUVEC), from pooleddonors (Lonza Cat #C2519A), passage 5-9, were cultured on 0.1% gelatincoated plates in EGM-2 endothelial cell growth media (Lonza). Cells weremaintained at 37° C. in a humidified atmosphere with 5% CO2. To measurecell proliferation, 1800 HUVECs suspended in 200 ul of endothelial basalgrowth media EBM-2 (Lonza) containing 0.5% PBS, were seeded in 96-wellplate. Four hours later recombinant Fc-fusion proteins and EYLEA atconcentrations of 10, 20, 50, 250, 500, 1000 and 2000 ng/ml were addedto cells along with 10 ng/ml of VEGF165. Cells were cultured for 3 days,and cell viability was determined by alamarBlue Cell viability reagent(Thermo Fisher Scientific), following the manufacturer's instruction.

In Vitro Binding to Bovine Vitreous

Bovine vitreous samples (InVision BioResource, Seattle, Wash.) werethawed at 4° C. and then diluted 1:1 with PBS, filtered through 0.22 pmfilter, aliquoted and stored at −80° C. Total protein concentrationswere measured by the Pierce BCA protein assay. Costar 96-well EIA/RIAstripwells were coated with vitreous (1 pg/well) for 4 hr at RT,followed by one wash with PBS-0.1% Tween 20 (PBS-T). To each well, 0.08to 10 nM chimeric VEGF receptor protein was added in a 50 pl volume andincubated overnight at 4° C. Plates were be then washed with PBS-T, andincubated with AP-conjugated goat anti-human Fc (1:2000, Invitrogen,#A18832) for 1 hr at RT. Plates were washed with PBS-T before 1 stepPNPP substrate (Thermo Scientific, Rockford, Ill., #37621) for 15-30 minat RT. Absorbance will be measured at 405 nm. S

Laser-Induced Choroidal Neovascularization (CNV)

Male C57BL/6J mice (6-8 week) were anesthetized with ketamine/Xylazinecocktail before laser treatment. CNV lesions were induced by laserphotocoagulation using a diode laser (IRIDEX, Oculight GL) and a slitlamp (Zeiss) with a spot size of 50 um, power of 180 mW and exposureduration of 100 ms.^(47, 65) Four laser burns were typically induced at3, 6, 9 and 12 o'clock position around the optic disc in each eye.Different constructs or IgG isotype control were injectedintravitreally, at the dose of 2.5 μg per eye, in a 1 pl volume. EYLEAwas used as a positive control at 2.5 or 25 μg. One day after injection,laser treatment was conducted and eyes were enucleated and fixed in 4%paraformaldehyde (PFA) for 15 min, 7 days after laser treatment. In aseparate set of studies, selected constructs were injected once 1 day, 7days or 14 days prior to laser treatment. Choroid-sclera complexes andretinas were separated and anti-CD31 immunofluorescence (IF) wasperformed to evidence the vasculature by whole mount staining of bothretina and choroidal tissues. For CD31 IF, rat anti-mouse antibody BD550274 was diluted 1:100 and incubated overnight at 4° C. After 4-hourincubation with a secondary anti-rat antibody (Life TechnologiesA11006), whole mounts were imaged at 488 nm. Quantification ofneovascularization in lesion area and vascular density in retina wascarried out by Image J. P values were assessed by Student's t test(significant change, p<0.05).

Oxygen-Induced Retinopathy Model

The Oxygen Induced Retinopathy (OIR) mouse model is a well-establishedmethod that has proven useful in delineating the molecular changes inischemic vascular eye disease⁶⁶ ⁶⁷ Using an enclosed chamber, neonatalmice are exposed to 75% oxygen from postnatal day 7 (P7) until P12, andthen returned to 21% oxygen (room air). This exposure to hyperoxiacauses vessel regression in the central retina and the cessation ofnormal radial vessel growth, mimicking the vaso-obliterative phase ofischemic vasculopathies. Upon return to room air, the avascular areas ofretina become hypoxic⁶⁸ ⁶⁹. This hypoxia induces the expression ofangiogenic factors, especially VEGF70, resulting in the growth ofaberrant retinal neovascularization at the junctions of vascular andavascular retina. To test the effects of inhibitors, intravitrealinjections will be performed prior to exposure to hyperoxia in an effortto test inhibition of the neovascular phase. Wild-type C57BL/6j mice atP7 will be anesthetized using isoflurane flowing through a rodentfacemask. The eyelids will be opened using a Vannas microdissectionscissors and pulled back to expose the eye. Next, 0.5 pl of solutionwill be injected using pulled glass micropipettes attached to apicospritzer III (Parker Hannifin) into the vitreous cavity. The needlewill be left in the eye for 30 seconds after injection and withdrawnslowly to minimize leakage. This procedure will be repeated in thefellow eye with injection of equimolar human IgG1 as control (Bio XCell, West Labanon, N.H.). EYLEA, various constructs will be testedversus control IgG1 at various doses. The eyelids were covered withantibiotic ointment. Litters will then be placed in a 75% hyperoxicchamber from P7-P12 to generate the OIR phenotype. At P17, the peak timefor neovascularization, the animals will be sacrificed, and the eyeswill be enucleated, dissected, and the vessels will be stained withBSL-FITC. The retinas were flat-mounted and imaged by confocalmicroscopy. The extent of neovascularization was quantified by measuringthe volume of pre-retinal vascular buds⁶⁷ ⁷⁰⁻⁷². Vaso-obliteration andneovascularization were analyzed using automated software, asdescribed⁷³.

CHO Cell Studies

Plasmid Construction and Expression

Nucleic acid fragments encoding extracellular Ig-like domains (ECDs) oneto three with the signal peptide of VEGFR1 (Gene ID: 2321) and a humanIgG1 Fc domains (Gene ID: 3500) were synthesized by GenScript USA Inc.The fragments were inserted into pD2535nt-HDP Dual EF1a-promoter vector(ATUM) at XbaI and ECoR1 sites, generating the plasmids expressing thefusion proteins of VEGFR1 ECDs with a 227-amino acid human IgG1-Fc. TheVEGFR1 ECDs constructs are as follows: V123 contains ECD1, 2 and 3;V1233 contains ECD1, 2, 3 and 3; V233 contains ECD 2, 3 and 3 and V23contains ECD 2 and 3. The authenticity of all constructs was verified bysequence analysis.

CHO K1 Glutamine Synthetase (GS) null cells (HD-BIOP3, Horizon) wereused for stable expression and transfections were carried out usingNeon™ Transfection System (#MPK10096, ThermoFisher). Briefly, linearizedconstruct DNA was transfected by electroporation into HD-BIOP3 cellsaccording to the protocol provided by Horizon Discovery, then the cellswere cultured in CD FortiCHO media (#A1148301, ThermoFisher) containing4 mM L-glutamine (#25030081, ThermoFisher) at 37° C. with humidifiedatmosphere of 5% CO₂ for 48 h. After the 2 day recovery, the media werechanged with selection media, CD FortiCHO containing 50 μM MSX (#76078,Sigma). For up to 20 days culture, four pools of VEGFR1 ECDs wereselected and banked. The expression of VEGFR1 fusion protein in theculture media was evaluated by human Fc ELISA Kit (EK000095-HUFC-2, SydLab Inc.) and western blotting with anti-human IgG1 Fc antibody(A-10648, Invitrogen). The expression levels for the four pools werefrom 1.9 to 13 μg per 1 million cells (7.1 for V123, 1.98 for V1233, 4.7for V233 and 12.7 for V23 in the average).

For single cell clone screening, the pool cells were diluted andselected according to the protocol (Horizon). After about 60 days'culture, total 39 clones, 8 for V123, 11 for V1233, 9 for V233 and 11for V23, were selected and stocked. The expression of VEGFR1 fusionprotein in the culture media for each clone was evaluated by ELISA andwestern blot. The expression level is from 3.0 to 18.3 μg per 1 millioncells (12 for V123, 3.7 for V1233, 6.5 for V233 and 13 for V23 in theaverage).

For large scale of culture media preparation, the cells (single cellclone) were seeded at the density of 0.5×10⁶/ml into the spinner flaskand cultured in the media of CD FortiCHO supplemented with 1:1000Anti-clumping agent (#0010057AE, ThermoFisher) and 1:200 ProteaseInhibitor Cocktail (p1860, Sigma) at 37° C. with 5% CO2 with humidifiedatmosphere and 125 rpm (Orbital shaker with a 25 mm orbit). Cellviability and density were monitored each day, and the culture mediawere collected after 5-7 days' incubation (the cell density is7.0-10×106/ml, viability is >90%). Clones, V1233-26, V233-52/67 andV23-5 were used for large culture media preparation. The expression ofVEGFR1 fusion proteins in the media was verified by ELISA and westernblot and media were stored at −80° C. for further purification. Theexpression level was from 20 to 115 μg/ml (23 μg/ml for V1233, 47 μg/mlfor V233 and 105 μg/ml for V23 in the average).

Purification

Horizon Discovery (HD)-BIOP3 CHO cells expressing higher levels of V1233protein (20-30 ug/ml) clones 14, 26, 44 and 46 were subjected topurification. All four clones yielded similar end product, thus forsubsequent purification we used clone 26. Condition media equivalent toroughly 10 mg from V1233-26 was performed as follows: Conditioned mediathawed at 37° C. was adjusted to 5% PBS and 0.01% (v/v) Tween20, and wascentrifuged at 20,000 g for 30 min at 4° C. The clarified extract wasapplied to a Protein A column (HiTrap™ MabSelect™Sure 5 ml) (GEHealthcare) equilibrated in 1×PBS and 0.01% Tween20. The column waswashed with high pH, high salt buffer (5 CV: 20 mM ethanolamine, pH 9.2,1.2 M Nacl, 0.01% Tween 20), and the bound proteins were eluted by 0.1Mcitric acid, pH 3.0, and were neutralized immediately by adding ⅕ thevolume of 1M Tris, pH 9.5. Fractions containing Flt1 protein werepooled, diluted 10× in 20 mM ethanolamine, pH 9.2, 0.01% Tween20, andapplied to HiTrap™Q HP 5 ml (GE Healthcare) anion exchange column. Flt1protein present in Flow through was adjusted to pH 6.8 by adding 10% v/v0.5M Tris, pH 6.8, and was applied to HiTrap™Heparin HP 1 ml columnequilibrated in 20 mM Tris, pH 6.8, 0.01% Tween20. The column was washedwith 0.45M NaCl in buffer, followed by final elution in 1M NaCl.Fractions positive for Flt1 were pooled, and was subjected to gelfiltration chromatography in HiLoad Superdex 16×600 column (GEHealthcare) in 10 mM Tris, pH 7.2, 0.4M NaCl, 0.01% Tween20. Fractionsexcluding the high molecular weight aggregates were pooled, concentratedafter binding to HiTrap™Heparin HP 1 ml column followed by 1M NaClelution as mentioned before.

The eluted proteins were dialyzed using Float-A-Lyzer®G2 dialysisDevice, MWCO 100 kD or 50 kD (Spectrum Laboratories), and concentratedby using Amicon centrifugal filters UltraCel 50k.

For large scale purification (condition media equivalent to 50 mgprotein), the method was modified as follows; Protein A chromatographywas done using HiTrapPrismA 5 ml column with two wash steps using buffer1 (50 mM Tris, pH 8.5, 1.2M NaCl, 0.5M Arginine, 0.01% Tween20) andbuffer 2 (25 mM sodium phosphate, pH 6.5, 200 mM NaCl, 0.01% Tween20)before final elution in 0.1M citric acid. HiTrapQ was performed using 20mM Tris, pH 8.5, and slightly higher salt (0.55M) was used to washHiTrap heparin column. Gel filtration step was performed in a widercolumn (HiLoad 26×600) in buffer 10 mM Histidine, pH 6.0, 80 mM NaCl,0.01% Tween20. Instead of dialysis, PD10 column was used for bufferexchange, and the final protein was stored in 10 mM sodium acetate, pH5.0, 7% trehalose and 0.01% Tween20.

Chromatography was carried out in FPLC system AKTA Avant (GEHealthcare). Column and the instrument were sanitized (cleaning inplace) by 0.5N NaOH before each run. Purity of the protein wasdetermined by SDS-PAGE and silver staining after each step. The qualityof final protein preparation was determined by analytical gelfiltration.

Total protein estimation was done by Protein assay dye reagent(Bio-Rad), and by Fc ELISA kit for human Fc proteins and human IgGs (SydLabs). Overall protein recovery was roughly 10%, and the final proteinachieved the level of endotoxin around 0.004 EU/mg, and HCP 150 ng/mg.

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What is claimed is:
 1. A method of treating a VEGF related condition inan eye of a subject comprising: administering to the eye an anti-VEGFagent consisting of the amino acid residues 27 to 459 of SEQ ID NO: 3.2. The method of claim 1, wherein the administering comprises anintravitreal injection.
 3. The method of claim 1, wherein the VEGFrelated condition comprises neovascularization.
 4. The method of claim3, wherein the neovascularization is choroidal neovascularization. 5.The method of claim 3, wherein the neovascularization is retinalneovascularization.
 6. The method of claim 3, wherein the VEGF relatedcondition is age-related macular degeneration.